This invention relates to an S. aureus specific mastitis assay.
Bovine mastitis is an inflammation of the bovine mammary gland or udder. While mastitis is most often caused by Staphylococcus aureus, it is also attributable to many other organisms including Streptococcus agalactiae, Pseudomonas spp., certain coliform bacteria and mycoplasmas. Mastitis damages the udder and lowers milk production, and therefore imposes an economic burden on the dairy industry. In view of the complex etiology of mastitis, the optimum treatment course may not be apparent until the organism is identified. This is typically done commercially by culturing the organism and classifying it by conventional taxonomic procedures.
Staphylococcus aureus is extremely complex from an immunological standpoint, and a variety of staphylococcal antigens have been studied as potential reagents in immunoassays for staphylococcal antibodies in milk or sera. These include antibodies against protein A, see Live and Ranu, J. Bacteriol., 96:14-23 (1968); enterotoxins, see Fey, et al., J. Clin. Microbiol., 19:34-38 (1984); hemolysins, see Spencer, et al., Am. J. Vet. Res., 24:83-98 (1963); Surujballi and Fackrell, J. Clin. Microbiol., 19:394-98 (1984); Opdebeeck, et al., Am. J. Vet. Res., 43:1770-75 (1982); Christensson, et al., Acta Path. Microbiol. Immunol. Scand., Sec B, 91:351-56 (1983); crude capsular antigens, see Opdebeeck and Norcross, Am. J. Vet. Res., 46:1561 (1985); Watson and Davies, Res, Vet. Sci. 1985, 39:52-58; whole bacteria, see Mathison, et al., Am. J. Vet. Res., 45:2518-24 (1984); teichoic acid, see Granstrom., J. Am. Microbiol., 17:640-46 (1983); peptidoglycan, see Christensson, et al., J. Clin. Microbiol., 19:680-86 (1984 ); leukocidin, See Loeffler and Norcross, Am. J. Vet. Res., 46:1728 (1985); and nucleases, see Gudding, Acta. Vet. Scand., 21:1-14 (1980).
Norcross and Obdebeeck, U.S. Pat. No. 4,425,330, used Staphylococcus aureus strain Wood 46 to produce a staphylococcal alpha hemolysin, which they crudely purified by the method of Coulter, J. Bacteriol., 92:1655-62 (1966). This preparation was then used as an ELISA reagent.
Our invention is distinguished from that of Norcross and Obdebeeck by the fact that it uses highly purified antigens with a molecular weight range of 18,000 to 26,000 daltons. The significance of their use as antigens is that virtually all S. aureus infected cows have antibodies in their milk which bind them and uninfected cows do not. The antigen preparation contains no alpha or beta hemolytic activity or significant quantities of polysaccharide.
The only commercial product that serves the same purpose as our invention is bacterial culture, the disadvantages of which are as follows: (1) it detects only live bacteria and therefore antibiotic residues may interfere with detection, (2) it is often inaccurate because of contamination and therefore requires that a sterile sample be obtained, (3) it is labor intensive and time consuming and (4) it costs about 10 times as much per sample as the present invention. There if no commercial ELISA for detection of antibodies in milk for any purpose that we are aware of.
The molecular weights of some of the staphylococcal protein antigens are as follows: alpha hemolysin (36,000), beta hemolysin (33,000), gamma hemolysin (45,000) leucocidin (31,000), enterotoxin A (34,700); enterotoxin B (28,366), enterotoxin C (34,100), enterotoxin C.sup.2 (34,000), enterotoxin E (29,600), enterotoxin F (20,000) and protein A (41,000). See Mollby, in Staphylococci and Staphylococcal Infections, at 644-645 (Easman and Adlam, eds: 1983), Vol. 2.